Glycosphingolipids (GSLs) play important roles in a wide variety of cell functions, including cell-cell interactions, cell growth and differentiation, and signal transduction. GSLs can interact with cholesterol to form membrane microdomains, and data from many studies suggest that the plasma membrane (PM) GSL and cholesterol composition may be tightly regulated. To achieve this regulation, cells must balance complex processes underlying the intracellular transport of GSLs with their synthesis and degradation. In the present application, four major projects will be pursued to better understand this regulation. We will (i) study the molecular determinants of GSLs that result in their selective endocytosis via a clathrinindependent, caveolar-like process in many cell types. Particular emphasis will be placed on the importance of lipid-lipid vs lipid-glycoprotein interactions at the PM in determining the internalization mechanism. We will also study the involvement of caveolin-1 in this endocytic process and the nature of GSL internalization in cells lacking caveolae; (ii) study the intracellular sorting of internalized SLs for recycling. Of particular interest is whether lipids internalized via coated pits recycle to the PM with similar rates as lipids internalized via caveolae, and whether they use the same protein machinery (e.g., rabs4 and 11). We will also investigate the distribution of GSL analogs into microdomains within early endosomes, and the rapid intermixing (and further sorting) of GSLs internalized via caveolae vs markers for the clathin pathway; (iii) test the hypothesis that PM GSL endocytosis and recycling are regulated by GSL synthesis and delivery to the PM, by modulating GSL synthesis or PM lipid composition; and (iv) continue our molecular and functional studies of glucosylceramide synthase which catalyzes the first glycosylation step in the formation of most higher-order GSLs, and is an important regulator of GSL homeostasis. The proposed studies are particularly timely because of many recent developments in the field pertaining to the organization of GSLs in membranes and to their emerging functional roles. A greater understanding of the caveolar-like endocytic pathway is also extremely important because the same or similar pathways are utilized for cell entry and intraceltular delivery of some bacterial toxins, viruses, and bacteria.